Among the number of different approaches for vaccine development, live bacterial vaccines are one of the most promising, as they mimic the route of entry of many pathogens and are able to elicit effective humoral and cellular immune responses, at the level of both systemic and mucosal compartments. Live bacterial vaccines can be administered orally or nasally, which offers advantages of simplicity and safety compared to parental administration. Batch preparation costs are relatively low and formulations of live bacterial vaccines show high stability. Attenuation can be accomplished by deletion of various genes, including virulence, regulatory, and metabolic genes.
Attenuated bacterial vaccines can not only be used to induce immunity to their corresponding pathogenic strain, but they can also be modified to deliver one or more heterologous antigens.
Attenuated derivatives of Salmonella enterica are attractive as vehicles for the delivery of heterologous antigens to the mammalian immune system because S. enterica strains can potentially be delivered via mucosal routes of immunization and have the ability to invade host tissues and persist, while continuing to produce a heterologous antigen. Furthermore, Salmonella strains elicit strong humoral and cellular immune responses, at the level of both systemic and mucosal compartments.
Several Salmonella typhimurium strains attenuated by aro mutations have been shown to be safe and effective delivery vehicles for heterologous antigens in animal models.
Approaches of delivering DNA constructs encoding heterologous antigens, in particular VEGF receptor proteins, via live attenuated Salmonella typhimurium strains into mouse target cells are described in WO 03/073995. Niethammer et al., (Nature Medicine 2002, 8(12), 1369) demonstrated that the attenuated S. typhimurium aroA strain SL7207 harboring an expression vector encoding the murine vascular endothelial growth factor receptor 2 (VEGFR-2 or FLK-1), which is essential for tumor angiogenesis, is functional as a cancer vaccine.
There is however only one attenuated Salmonella enterica serovar strain, namely Salmonella enterica serovar typhi Ty21a (short: S. typhi Ty21a), which has been accepted for use in humans.
This well-tolerated, live oral vaccine against typhoid fever was derived by chemical mutagenesis of the wild-type virulent bacterial isolate S. typhi Ty2 and harbors a loss-of-function mutation in the galE gene, as well as other less defined mutations. It has been licensed as typhoid vaccine in many countries after it was shown to be efficacious and safe in field trials.
There is a strong demand for live attenuated bacterial vectors as delivery vehicles for heterologous antigens—especially cancer antigens—that are safe for use in humans. The provision of such an attenuated bacterial vector as DNA vaccine also calls for the efficient, high-yield cultivation of the attenuated bacterial strain transformed with said heterologous antigen DNA. Transformation of bacterial strains with recombinant DNA constructs often results in decreased cell growth. Thus, it is often necessary to improve the preferably large-scale cultivation process to obtain high yields of viable and functionally active cells.